Cells contain thousands of protein “micromachines” performing a bewildering number of chemical reactions every second. The challenge for biologists in the 21st century is to integrate information about multiple – or even all – proteins into holistic models for the entire cell. This is a daunting task. The addition of any new component to a system can alter the behavior of the components already there. This phenomenon is especially familiar to biologists studying the cytoskeleton, a complex system of protein filaments that provide the force for cell division and migration, among other things. The building blocks of the cytoskeleton are simple proteins called tubulin and actin that assemble into a remarkable variety of shapes depending on context. While the basic chemistry of this assembly process has been understood in purified systems for decades, how it happens in cells is not well understood. For example, growth of actin filaments is a two-step process: nucleation, or the formation of a new filament, and elongation, or the extension of that existing filament. Both steps happen just fine when actin is present in pure form in a test tube. In cells, however, proteins called profilin and capping protein block these two steps, respectively. Nucleation and elongation can only occur because other proteins overcome these blocks. Thus, a faithful experimental system to study actin assembly as it would occur in a living cell requires – at a minimum – five purified proteins.

One technological advance of great importance is the ability to literally see single molecules (in this case proteins) using advanced fluorescence imaging. In such an experimental system, many details can be captured. In a recent publication in Science, Dr. Dennis Breitsprecher and colleagues in the Goode and Gelles labs, undertook this challenge and directly visualized the effects of key regulatory proteins helping actin proteins nucleate and grow into filaments in the presence of both profilin and capping protein. A previous study from the Goode lab had shown that two proteins, called APC and mDia1, together stimulate the growth of actin into filaments (Okada et al, 2010). In the present study, Breitsprecher and colleagues examined the mechanism by which APC and mDia1 overcome the profilin and capping protein-imposed blocks. To do this, they ‘tagged’ actin, APC and mDia1 with three different fluorescent dyes, each of a different color, and then filmed these molecules (using triple-color TIRF microscopy) in the act of building an actin filament to learn precisely what they are doing.

The authors began by imaging APC and actin (2 colors) at the same time. APC formed discrete spots on the microscope slide, and growing actin filaments emerged from them, suggesting than APC nucleates actin filament formation. As the filament emerged from the APC spot, APC stayed where it was: remaining stably associated at the site of nucleation. Next, the authors added dye-labeled mDia1 to the system, and observed mDia1 molecules staying attached to and ‘riding’ the fast-growing end of actin filament, while protecting it from capping protein.

The most remarkable result came when they visualized all three labeled molecules together (actin, APC, and mDia1). What they saw was that APC and mDia1 first come together in a stable complex even before actin arrives. Then APC recruits multiple actin subunits to initiate the nucleation of a filament. This complex was resistant to the blocks imposed by both profilin and capping protein. As the filament grew from the APC-mDia1 spot, mDia1 separated from APC and stayed bound to the growing end of the filament – protecting it from capping protein while it grows. Thus, even though APC and mDia1 have different activities, participating in different stages of the growth of a filament, they associate together before actin even arrives, likely so that once the actin filament is born, it is immediately protected from capping protein. This mechanism has been compared to a rocket launcher: APC is the launch pad for an actin filament, which is then propelled forward by mDia1.

The new study provides great detail of the system: for example, the number of APC subunits required to nucleate actin filaments was determined, and the growth rate of actin filaments in the presence and absence of all the other components was measured. Ultimately, all of these data will be required to put together a detailed model of how actin filaments grow inside of real cells: details that would be difficult or impossible to obtain without employing single molecule analysis.

For the future, the authors have set their sights on even more challenging experiments aimed at elucidating the mysterious link between tubulin and actin fibers. APC and mDia1 are implicated in this linkage in living cells, but almost nothing is known about how they physically link and/or communicate information between the two systems. Since APC is mutated in some 80% of colon cancer tumors, understanding its multiple roles is of clinical as well as intellectual importance. This will be an exciting, if challenging, endeavor for the future.

Single-molecule fluorescence imaging is often incompatible with physiological protein concentrations, as fluorescence background overwhelms an individual molecule’s signal. Loveland et al. employ a new imaging approach called PhADE (photoactivation, diffusion and excitation). A protein of interest is fused to a photoactivatable protein (mKikGR) and introduced to its surface-immobilized substrate. After photoactivation of mKikGR near the surface, rapid diffusion of the unbound mKikGR fusion out of the detection volume eliminates background fluorescence, whereupon the bound molecules are imaged. The authors labeled the eukaryotic DNA replication protein flap endonuclease 1 with mKikGR and added it to replication-competent Xenopus laevis egg extracts. PhADE imaging of high concentrations of the fusion construct revealed its dynamics and micrometer-scale movements on individual, replicating DNA molecules. Because PhADE imaging is in principle compatible with any photoactivatable fluorophore, it should have broad applicability in revealing single-molecule dynamics and stoichiometry of macromolecular protein complexes at previously inaccessible fluorophore concentrations.

This past June the MRSEC Center offered a condensed summer course based on the popular graduate course QB120: Quantitative Biology Instrumentation Laboratory.

The course was taught by Zvonimir Dogic of the Physics Department (pictured). Prof. Dogic has extensive experience with several forms of microscopy and his Lab features several home-built or heavily modified optical setups.

The course is designed to offer students hands on experience with building their own optical setups from basic components as well as learning how to optimally acquire imaging data from commercial microscopes. The focus was on understanding the physics behind microscope function and leveraging that knowledge towards improving data acquisition in the lab.

Initially, students used basic lenses, apertures, an objective, a camera and a light source to build the simplest possible light microscope. This initial setup was quickly extended to include Köhler illumination, a core principle in microscopy which allows even illumination of the sample as well as access to the conjugate image plane for image filtering.

The next project required students to build a fluorescence microscope, a highly relevant and ubiquitous technique in biological imaging. To image a slide with fluorescently labeled beads students used a dichroic mirror to separate excitation light at one wavelength from emission light at another wavelength. A schematic diagram, a photo of this setup with the light path superimposed and actual data acquired with one of these microscopes can be seen in the video below.

Next, a more advanced technique in microscopy, total internal reflection microscopy (TIRF), was introduced and an imaging setup using this technique was built. TIRF microscopes excel at imaging small molecules that are immobilized in a small area. A laser beam was pointed to shine through a prism at an angle sufficient to cause total internal reflection and the resulting evanescent wave caused fluorescent excitation of the sample. The video below shows a schematic and imaging data of a TIRF microscope built by students.

Finally, students used commercial microscopes to understand the principles behind phase contrast and difference interference contrast microscopy, both techniques well suited for imaging samples that are nearly transparent.

Overall the Course provided an excellent introduction to the physical principles behind microscope function. I highly recommend it to anyone interested in using microscopes in their research!